Insectes Sociauz, Paris. 1974, Vol. 21, n" 1, pp. 87-94.

LABORATORY INVESTIGATIONS OF THE TRAIL-FOLLOWING RESPONSES OF FOUR SPECIES OF LEAF-CUTTING WITH NOTE! ON THE SPECIFICITY OF A TRAIL OF ATTA TEXANA (BUCKLEY)

By S. W. ROBINSON *, J. C. MOSER **, M. S. BLUM *** and E. AMANTE **** " Department of Applied Zoology, Uniuersify College of North Wales, Bangor, Caerns., U.K "" Southern Forest Esperimenf Sfation, Pineville, Louisiana 71360, U.S.A., *** Ljeparfmenf of Enfomology, Universify of Georgia, Athens, Georgia 30602, C.S.A. and """* Znstituto Bioldgico, Caixa Postal 7119, SGo Paulo, Brazil.

Rep le 2 janvier 1974. Acceptk le 23 fkvrier 2971

SUMMARY

Interspecific trail following activity of poison sac contents from four species c leaf-cutting ants was investigated. With only one exception, all the species teste followed trails made from each others poison sac contents. However when the an1 were given a choice of following one of two separate trails, clear differences were show in the poison sac contents of the three species tested. The responses of 24 species of ants and one species of termite to an Atta fexan (Buckley) trail pheromone were tested.

Etude au Jaboratoire de reponses mufuelles aux pistes chez quafre especes de Fourmis Atta. Spdcificifh d%ne phdromone de piste de Atta texana (Buckley).

Xous avons Ctudii? comment quatre especes de Fourmis ifttn suivent mutuelleme~ les pistes trackes avec le contenu de leur sac B venin. Toutes les especes PtudiPes or suivi les pistes des autres B une seule exception. Cependant, lorsque nous avons donr aux Fourmis le choix entre deux pistes distinctes, nous avons montrk qu'il y ara des diff6rences dans le contenu du sac B venin de trois esphces. Kous avons CtudiC les rCponses de 24 especes de Fourmis et d'une esphce de Termil B une pheromone de piste de Affa fesana (Buckley). 90 S. W, ROBZXSON, J. C, MOSER, kf. S. BLUM AND E. ALWAAc'TE:

TABLEI. - The numbers of Q: lost s ants (out of a total of 40 from four replicate expe. rimenfs) that followed poison sac trails.

TABLEAUI. - Xombre de Fournis G perdues B (sur un total de $0 dans qtintre exptrience: identiques) qui suivirent les pistes tracdes avec fe contenu du sac a uenin.

Pheromone donor Test species A cronzyrmex Afta At ta Atfn octospit~osrrs cephalofes sexdens texa~za Acrom yrrnex octospinosus ...... 28 2 3 34 39 A tta cephalofes ...... 2 8 40 34 38 ...... 0 3 6 34 3 3 Atta fexana ...... 17 20 20 19

11. - Response to a choice of trails.

A second experiment was done to determine if the ants could distinguish between trails made from poison sacs of their own species and trails made from poison sacs of other species that they would nonetheless follow. The results are shown in Table 11.

TABLE11, - The numbers of ants follozving e: poison sac >> trails when given a choice of two alternatives. All individual results are pooled from eight separate tests.

TABLEAU11. - hTombre de Fourmis gui ont suivi les pistes tracCes avec le conten11 dzi a sac d venin >>, Iorsqu'elles ont eu le choix entre deux solzrtions possibles. il'orrs avons rassembld les re'srzltats de huit expe'riences. Pheromone donors Test species '4.0. : Ax. A.s. : A.c. A.o. : A.s. Acromyrmex octospinosus ...... 42 : 19 - 57 : 11 (p <0.01) ip <0.001) Atta cephalotes ...... 40 : 98 73 : 62 - (p <0.001) (p >0.3) " Atta sexdens ...... - 93 : 48 - (p <0.001) - : not tested. The probabilities shown are calculated from pooled Chi squared values assuming a 1 : 1 ratio. " Replicates showed significant heterogeneity.

In all cases, but one, the test species followed trails from poison sacs of its own species in preference to those from sacs of other species, the exception being Atta cephalotes which showed no significant preference between its own poison sac contents and those of Atta sezdens. Afta cephaloies was used for further experiments using a similar teclinique. Foraging workers were given a choice of following trails made from poison sacs of ants from the same colony (a own B) and trails made from sacs removed from ants of a different colony (a strange B). The pooled resr-tlts from eight replicates gave an a own >> followed to strange >> followed ratio of 96 : 91 (the pooled Chi squared value based on 8 replicates with an expected ratio of 1 : 1 = 0.13 with 1 d.f. p >0.7 replicates were hon~ogenous). A similar experiment was also done with natural trails by using string on which the ants had been recently foraging. The pooled resrilts gave an (I: own >> : :strange B ratio of 96 : 87 (the pooled Chi squared value based on 10 replicates with an expected ratio of I : 1 = 0.44 with 1 d,f. p >0.6 replicates showed significant heterogeneity). To ensure that the foraging ants were iiiaking a choice at the << Y >> junction a treated string was cornpared with an untreated string. The pooled results gave a treated 3 : a untreated >> ratio of 25 : 5 (the pooled Chi squared value based on 2 replicates with an expected ratio of 1 : 1 =: 13.33 with 1 d.f. p <0.001. Replicates were homogenous).

111. - Bioassay of methyl-4-methylpyrrole-2-carboxylate.

M4kIP2C was tested for trail following activity with a total of 24 species froiii several subfanlilies of ants and on one species of termite. The responses of the ants are shown in Table 111. Table I11 shows that in general, Attines will follow trails of h'14MP2C between the ranges of 4.0 pg to 4.0 x 104 pg spread around a 50 cm circular trail, the only exception was Atta sezdens which could not be made to follow a M4MP2C trail at any of the concentrations tested. Non-attine species did not follow trails streaked with M4MP2C. The termite Reticulitermes flauipes (ICollar) did not follow a trail of 314MP2C.

DISCUSSION AND CONCLUSIONS

Several workers have shown in the laboratory that attine trails prepared from poison sac estracts are not species or even genera specific. However, responses of ants to artificial trails in the laboratory cannot always be correlated uTitli field observations, and field studies indicate that the trails of leaf cutting ants appear to be specific. In field studies with Atta cephalotes isthmicofa (IVeber) and Atta colontltica tonsipes (Santchi), \VEBER (1969) demonstrated that workers of both species when placed onto the trail of the other species, recognised the odour trait, became excited, and quickly left it. BLUMet al. (1964) also noted in two field populations of leaf cutting ants that emit. Acromyrmex octospinosus workers when placed on an Atta cephalotes trail, would invariability leave the trail and resume trail following only when it crossed an A. octospinoszrs trail. They also recorded that although active field trails of A. cephalotes and A. ocfospi- nosus crossed in at least two places, in no instances were the ants seen to follow the trail of the other species. They could not duplicate this behaviour in the labo- ratory using poison sac estracts and attributed it to cornpounds other than trail substances (e.g. anal products). IIowever BLUM and P~RTOCARRERO(1966) TABLE111, - The nrrnzbers of anfs responding to $0 ul of ucxriozrs concenfrr~tions M$A%fP2Ctrailed nround a 54 em circtrntference circular trail.

TABLEAX'111. - Sombre de Fournris nyant re'pondzr ci 10 Cil de sc_ifzzfionsdc .tfiL%fIJ2 des concenfrafiorzs uarie'es, repandzles selon rrne pisfe eircufaire cfe 50 crrz de diantl

Attini : '!Affa fexana (Bucliley) ...... O 0 13 17 19 13 3 0 - 'dtta sexderzs (I,.) ...... 0 0' 0 0 0 0 0 0" fAtta sesdens ssp. rrrbropilosa Forel . . 0 0 0 3 110 1 ?Atfa cephalofes (IA.) ...... 0 1 11 lfi 10 5 1 1 'IAftu colonlbica Guerin ...... 0- 113 li 1 0 0- fAtfa laeuigafa (F. Smith)...... 003O57OO "* bAcromyrmes octospinosrzs (Reich) . . 0 0 22 15 56 29 11 0 fdcronzyrmes niger (F. Smith)...... 0 0 0 1 0 0 0 0 riAcromyrnzex uersicolozzr I'ergande. ... 0 0 6 10 I ti 10 5 0 - "Trach!]m;jr~nexsep ferr frioncrlis ihlcCook) ...... "Trachgmyrmex nriclzi (Forel) ...... hApterosti!~macollul-e (Emery) ...... ~'f:gphornyrrnezrirnosrrs (Spinolaj ..... Non-attine species : "'Cardiocondyla uenrrstlrlcx \fT.;1I.Wheeler "Pheidole denfata Jlayr...... "Solenopsis inuicfa I3uren ...... l'Pogo~zornyrnzex hurbtrtr~sF. Smith. ... 'iCrenzato~/crsterclara hlayr...... ',l.leiuumyrntex rt i~jrescerzsiCrcsson) .... 'Formica rtrfa (I,.) ...... "l'trratrechina parunln (Alayr) ...... "(:crrnponotus pennsyluarziclrs (1)eGreev). Iridom yrnzex prrr inosrrs (I'loger) ...... t"Psenclomyrnzex Drunnee (I;. Smith). ...

a : Bio:issaved by 31. .hf. TA ART IN. b : Bioassa:\.ed by 51. S. I31,r.s~and identified hy .S. A. WESER. c : Bioassayed by J. 17. WATKIXS11. d : Bioassayed by J. C. MOSER. e : Bioassayed hy S. W. Roe~xsori. f : Uioassayed by E. AMANTE. g : 20 ants tested. h : 30 ants tested. i : I tested. - : not tested.

showed that not all Attine poison sac contents are identical by dcl~lonstratingtl Sericort~yrnlexurichi will not follour an dtta texana trail and will only weal follow a trail prepared froill a Trachgrttymzex septentriortalis poison sac.

When (i lost >> ants from the four species were tested on a circular tr produced from poison sac extracts of the other species, most ants readily follow ATTA TEXASA ( BUCKLEI") 93 the trail, the only exception being Atta sexdens which would not follow a trail frorti an Acronzyrnzex octospirzostzs poison sac. This is somewhat surprising as the trail foliotving response of A, oetospinosrrs to A. sexderzs poison sacs is strong. l3~v~and Ross (1965) demonstrated that trails prepared from poison sac extracts of 11iyr1nicines in unrelated genera cotlld be non specific even at the generic level. They suggested that it was not inlprobable that nlyrnlicines might have a nunlber of conltilon cheniical features in their venoril and that even tho~~gflants fro111 different genera ntigllt readily follow trails rnacte fro111 each others poison sacs extracts, they may each be enlploying different conlpounds for their oclour trail plteronlone. It seerris that the conipound that dfta sexderzs utilizes for trail following is not found in Acronzyrnzex octospirzosrzs poison sacs, although the conrpound l~yA. octospirzosrzs is present in the A, sexdens poison sac but not used as a trail marker by A. sexdens. This would allow A. octospi~zosr~s to follow trails ~iiadefro111 A. sexderzs yoison sacs but not vice-versa. ?J'ATKIXSet ul. (1967) using various species of army ants dernonstrated that in nearly all cases, army ants would readily follow trails rilatfe by workers of other army ant species. IJ'llen they were given a clloice of either of two trails to follow, all the species tested (with one possible exception) chose to follow a trail laid by a rllerilber of its ow11 species. Siinilar results were obtained in the present trials with the attines. Altllougl~ in general attines will readily follow trails rliade fro111 poison sacs of other attines, the results of the trail preference tests (table 11) sllour that differences in poison sac contents do exist both between and within genera. In only one experiment, when A. cephalotes was given a choice of a trail made froni its own or an A. sexderzs poison sac, was there evide~iceof a species being unable to distinguish between tile two trails. However when the same choice was tested with A. sexdens a clear preference was shown. The results for A. cephalotes do not preclude the possibility of A. cephalotes being able to recognise these differences in the field where both these species may be found in the sarlte habitat. The abilities of the ants to distinguish between the poison sac contents of various species in the same genus can only be demonstrated by the inore sensitive trail choice experiments described. As table 111 derrlonstrates the volatile component of the trail secretion of the texas leaf cutting ant Atta texa~za(314h11'2C) was tested on 24 species of ants fro111 several tribes ancl on one trail laying termite. No non-attines followed a 314hfP2C trail, although all the attines tested, with the exception of Atta sexdens, did. As table 1 sl~o?\~s,Atta sexdens together with A. cephalotes and Acromijrrnex octospinostrs will readily follow trails illade fro111 u~holeA. texcir7a poison sacs, and this again suggests that the Attines are not all using the same compounds for trail ntarking. Atta sexderzs is clearly either responding to some other conlpounds in the A. texana poison sac or 11'ill only follow trails of MithfP2C when some otlrer corllpounds are also present. Although Aftn sexdens could not be made to follou- a 314?iIF'2C trail, Ro~rssosand CHERRETT (1973) have shown that 3I4JII)2C is an etfective attractant in baits for A. sexderzs as well as for A. cepha- lotes and A. octospinosrzs. Thus it appears that at least some of the Attines utilize, for the purpost trail following, different trace constituents in their venom. TUMLIKSO~;et a!. (19723, whilst assaying fractions of the volatile componc of an Afta texana extract, found that more than one fraction was active promoting trail following althougfl larger quantities of the fractions wit1 M4MP2C were needed. It is these minor active components that may a1 leaf-cutting ants to make a choices >> when given an option of two trails possibly one of these that Attu sexdens relies on particularly for marking following trails.

ACKNOWLEDGEMENTS.- Part of the studies were financed by n grant from. Overseas Development Administration of the British Foreign and Commonwealth Of

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